Expanding Glycomic Investigations through Thiol-Derivatized Glycans

N-(2-thioethyl)-2-aminobenzamide (TEAB), a novel glycan auxiliary, was synthesized and its utility was evaluated. The auxiliary was conjugated to glycans by reductive amination with the water-stable reagent 2-picoline borane complex. Glycan products, which ranged from 1 to 7 linked hexoses, were all isolated in yields ranging from 60% to 90% after purification by reverse-phase chromatography. The novel conjugate introduces a convenient, shelf-stable thiol directly onto the desired free glycans with purification advantages and direct modification with efficient reactions through alkenes, halides, epoxides, disulfides, and carboxylates in yields of 49% to 93%. Subsequently, a thiol-selective modification of the BSA protein was used to generate a neoglycoprotein with a bifunctional PEG–maleimide linker. To further illustrate the utility of a thiol motif, 2-thiopyridine activation of a thiol-containing support facilitated the covalent chromatographic purification of labeled glycans in yields up to 63%. Finally, initial proof of concept of implementation in a light printed microarray was explored and validated through FITC-labeled concanavalin A binding. In conclusion, the thiol-functionalized glycans produced greatly expand the diversity of bioconjugation tools that can be developed with glycans and enable a variety of biological investigations.


Introduction
Glycomics has been of growing interest within the scientific community recently but has historically [1][2][3] lagged behind proteomics and genomics due to the inherent difficulties of isolation and subsequent application of complex glycans. Glycomics investigations commonly rely on sophisticated techniques such as hydrophilic interaction chromatography (HILIC), polymeric anion exchange (WAX), and porous graphitic carbon (PGC) to purify complex glycans [4,5] and mass spectrometry (MS) [6] to detect desired glycan-containing fractions, and it is further challenging to perform chemical modifications of the selected glycans to facilitate biological studies [7]. In contrast, the field of proteomics has brought about innovative [8], selective, mild [9], and occasionally reversible modifications [10] of proteins enabling site-specific tagging [11] and pull-down approaches [12]. Therefore, the field of proteomics has rapidly progressed with assigning structures and functions to an ever-increasing number of targets [13]. Many of these chemical biology approaches exploit thiol chemistry, including intrinsically present cysteine residues, due to the soft nucleophilic nature of thiol groups [14][15][16].
Glycans containing reducing ends are typically conjugated with an aromatic auxiliary using reductive amination, where the reducing end of the glycan reversibly forms an aldehyde as the ring opens, allowing for reactivity with an amine of interest to form an intermediate imine, which is then reduced with a borane reagent to form the secondary amine-labeled glycan [6]. This then allows for UV-visible (UV-Vis) detection and efficient separations using high-performance liquid chromatography (HPLC). The auxiliary 2-amino-N-(2-aminoethyl)-benzamide (AEAB) has become the most used auxiliary after being popularized by Cummings and Song [17]. AEAB-functionalized glycans after reductive amination possess a primary amine to further conjugate to a handful of functional groups such as N-hydroxysuccinimide (NHS) esters and epoxide-linked solid supports or are converted to azides to enable Staudinger ligation [18][19][20][21][22]. Current glycan auxiliaries, shown in Figure 1, include 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), anthranilic acid (AA), and aminobenzamide (AB) and have also been utilized extensively [23] but possess an even more limited bifunctional arm than is present in AEAB. Many of these glycan auxiliaries have been shown to not separate via traditional C18 chromatography and instead must use elaborate techniques such as HILIC/PGC or ion-pairing chromatography to be isolated from crude reaction mixtures, which can vary greatly depending on solvent conditions and the type of column, therefore limiting scalability and reproducibility [5]. Previous studies have also evaluated these synthetically modified glycans as coupling partners to various enzymes and shown general binding to glycosyl transferases [24][25][26] to generate complex glycans as well as posing general binding to most lectins [27]. To further expand upon the success of the bifunctionality of the aromatic reporter, a thiol-containing group was evaluated as a versatile tool to increase the functionality of labeled glycans.
amine-labeled glycan [6]. This then allows for UV-visible (UV-Vis) detection and efficient separations using high-performance liquid chromatography (HPLC). The auxiliary 2amino-N-(2-aminoethyl)-benzamide (AEAB) has become the most used auxiliary after being popularized by Cummings and Song [17]. AEAB-functionalized glycans after reductive amination possess a primary amine to further conjugate to a handful of functional groups such as N-hydroxysuccinimide (NHS) esters and epoxide-linked solid supports or are converted to azides to enable Staudinger ligation [18][19][20][21][22]. Current glycan auxiliaries, shown in Figure 1, include 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), anthranilic acid (AA), and aminobenzamide (AB) and have also been utilized extensively [23] but possess an even more limited bifunctional arm than is present in AEAB. Many of these glycan auxiliaries have been shown to not separate via traditional C18 chromatography and instead must use elaborate techniques such as HILIC/PGC or ion-pairing chromatography to be isolated from crude reaction mixtures, which can vary greatly depending on solvent conditions and the type of column, therefore limiting scalability and reproducibility [5]. Previous studies have also evaluated these synthetically modified glycans as coupling partners to various enzymes and shown general binding to glycosyl transferases [24][25][26] to generate complex glycans as well as posing general binding to most lectins [27]. To further expand upon the success of the bifunctionality of the aromatic reporter, a thiolcontaining group was evaluated as a versatile tool to increase the functionality of labeled glycans.

Results
The synthesis of N-(2-thioethyl)-2-aminobenzamide (TEAB) was previously described by Sherman and Beck [28] utilizing anthranilic acid, cysteamine dihydrochloride (1) with hydroxybenzotriazole (HOBt), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), followed by NaBH4-mediated reduction and finally purification with silica gel chromatography. In order to establish a more efficient and scalable synthesis of TEAB, an alternative approach using the activated carbonyl isatoic anhydride (2) was investigated [29]. Nucleophilic acyl substitution and decarboxylation of 2 proceeded readily in the presence of diisopropylethylamine and a substoichiometric amount of 4-dimethylaminopyridine (DMAP) overnight at 50 °C. The resulting disulfide (3) precipitated from the aqueous solvent as the reaction proceeded, providing the product in good yield and purity after sufficient washing of the desired solid. Reduction of disulfide 3 with zinc powder [30] furnished the thiolate-zinc salt 5 (Zn-TEAB), which could be isolated by precipitation in 94% yield (displayed in Figure 2). Alternatively, tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 eq.) could be employed to reduce the disulfide 3 to the thiol 4 (TEAB) in 37% yield. TEAB (4) was observed as an excellent metal chelator when the thiol form was isolated, resulting in oxidation to disulfide 3 occurring even when dissolved in deionized water. Consequently, thiol salt 5 is a shelf-stable source of thiol (3) that does not readily oxidize as a solid or in solution over two months (results displayed in supplementary materials) as well as possessing similar fluorescent properties comparable to other popular aminobenzamide derivatives due to the unchanged electronics present on the aromatic ring.

Results
The synthesis of N-(2-thioethyl)-2-aminobenzamide (TEAB) was previously described by Sherman and Beck [28] utilizing anthranilic acid, cysteamine dihydrochloride (1) with hydroxybenzotriazole (HOBt), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), followed by NaBH 4 -mediated reduction and finally purification with silica gel chromatography. In order to establish a more efficient and scalable synthesis of TEAB, an alternative approach using the activated carbonyl isatoic anhydride (2) was investigated [29]. Nucleophilic acyl substitution and decarboxylation of 2 proceeded readily in the presence of diisopropylethylamine and a substoichiometric amount of 4dimethylaminopyridine (DMAP) overnight at 50 • C. The resulting disulfide (3) precipitated from the aqueous solvent as the reaction proceeded, providing the product in good yield and purity after sufficient washing of the desired solid. Reduction of disulfide 3 with zinc powder [30] furnished the thiolate-zinc salt 5 (Zn-TEAB), which could be isolated by precipitation in 94% yield (displayed in Figure 2). Alternatively, tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 eq.) could be employed to reduce the disulfide 3 to the thiol 4 (TEAB) in 37% yield. TEAB (4) was observed as an excellent metal chelator when the thiol form was isolated, resulting in oxidation to disulfide 3 occurring even when dissolved in deionized water. Consequently, thiol salt 5 is a shelf-stable source of thiol (3) that does not readily oxidize as a solid or in solution over two months (results displayed in Supplementary Materials) as well as possessing similar fluorescent properties comparable to other popular aminobenzamide derivatives due to the unchanged electronics present on the aromatic ring.
With gram quantities of Zn-TEAB (5) in hand, conjugation to a glycan substrate was evaluated. Typically, reductive amination is conducted using sodium cyanoborohydride as a reducing agent to reduce the intermediate imine [31], but it was deemed more advantageous to utilize the 2-picoline borane complex [32] due to its water-stability and relatively non-toxic properties, therefore allowing for faster glycan conjugation and less hazardous reaction conditions when scaled up. Ethylenediaminetetraacetic acid (EDTA) was also included in the reaction in excess to prevent thiol oxidation and to coordinate the zinc ion. Once completed, the crude mixture was filtered and TCEP (2.5 eq.) was added to ensure isolation of only thiol-conjugated glycans. The mixture was concentrated and purified by preparative reverse-phase chromatography using a gradient of 99:1% to 50:50% (water:acetonitrile) to provide the desired products, shown in Figure 3 (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). This process is unique to TEAB, as other major auxiliaries such as AEAB/AB/AA and ANTS require extensive purification via HILIC/ion-pairing chromatography since these auxiliaries do not enable retention to C18 media. The co-solvent mixture chosen for the reaction was a compromise from a simple water:acetic acid mixture to allow for the proper solvation of the Zn-TEAB auxiliary whilst still solvating the longer glycans of interest, such as maltoheptaose. With gram quantities of Zn-TEAB (5) in hand, conjugation to a glycan substrate was evaluated. Typically, reductive amination is conducted using sodium cyanoborohydride as a reducing agent to reduce the intermediate imine [31], but it was deemed more advantageous to utilize the 2-picoline borane complex [32] due to its water-stability and relatively non-toxic properties, therefore allowing for faster glycan conjugation and less hazardous reaction conditions when scaled up. Ethylenediaminetetraacetic acid (EDTA) was also included in the reaction in excess to prevent thiol oxidation and to coordinate the zinc ion. Once completed, the crude mixture was filtered and TCEP (2.5 eq.) was added to ensure isolation of only thiol-conjugated glycans. The mixture was concentrated and purified by preparative reverse-phase chromatography using a gradient of 99:1% to 50:50% (water:acetonitrile) to provide the desired products, shown in Figure 3 (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). This process is unique to TEAB, as other major auxiliaries such as AEAB/AB/AA and ANTS require extensive purification via HILIC/ion-pairing chromatography since these auxiliaries do not enable retention to C18 media. The co-solvent mixture chosen for the reaction was a compromise from a simple water:acetic acid mixture to allow for the proper solvation of the Zn-TEAB auxiliary whilst still solvating the longer glycans of interest, such as maltoheptaose.  A variety of commercially available glycans were selected with varying chain lengths of hexoses and stereochemistry present between all substrates. The yields of the resulting glycoconjugates generally reflected the ease in isolation, with disaccharides such as lactose achieving a 90% yield while the tetrasaccharide acarbose achieved a poorer yield of 60%. Reactivity between the substrates was similar, with no further reductive amination observed past 2.5 h. In addition, uniformity between all separations was observed to be A variety of commercially available glycans were selected with varying chain lengths of hexoses and stereochemistry present between all substrates. The yields of the resulting glycoconjugates generally reflected the ease in isolation, with disaccharides such as lactose achieving a 90% yield while the tetrasaccharide acarbose achieved a poorer yield of 60%. Reactivity between the substrates was similar, with no further reductive amination observed past 2.5 h. In addition, uniformity between all separations was observed to be significant, with only a 30-second change in retention time during reverse-phase chromatography from xylose to lactose and a further 30 s to maltoheptaose. In the purification, small single glycans were challenging to separate from starting materials as they eluted a full minute closer to TEAB (4), while the longer glycans retained slightly less to the C18 media. Finally, inclusion of TCEP resulted in poorer yields of the larger glycans, causing more co-elution of the glycoconjugates (16 and 17) at the dead volume in preparative C18. The overall yields are comparable to other auxiliaries utilized but with the significant difference of the utilization of C18 chromatography instead of the less reproducible HILIC/ionpairing chromatography.
With the TEAB glycan conjugate probes ready, the chemoselective conjugation of the thiol-derivatized glycans was investigated. Lactose-TEAB (14) was selected to identify the optimal conditions for reaction with a diverse range of functional groups ( Figure 4). To display the utility of a thiol group over an amino derivative, a wide variety of coupling partners were selected to highlight the selective chemistry of thiols.  First, conjugate addition to the maleimide functional group was performed in the presence of a volatile base, (NH4)2CO3, to promote generation of the thiolate ion to attack the maleimide present. Addition was achieved in 30 min, yielding the desired product 18, which was then purified from trace salts via prep-C18 in 76% yield. Next, thiol-ene click addition to endo-norbornene-cis-5,6-dicarboxylic acid was performed with the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.05% w/v), which proceeded in an exo-selective manner, resulting in product 19. The desired product was purified with prep-C18 in 93% yield, and stereochemistry was indicated by 2D NOESY and HSQC NMR (supplied in supporting information, SI 25-27) and supported by the literature [33]. Asymmetric disulfide synthesis of 20 is a reversible conjugation to lactose-TEAB, where an unstable disulfide bridge is formed by an excess of dipyridyl disulfide reagent. First, conjugate addition to the maleimide functional group was performed in the presence of a volatile base, (NH 4 ) 2 CO 3 , to promote generation of the thiolate ion to attack the maleimide present. Addition was achieved in 30 min, yielding the desired product 18, which was then purified from trace salts via prep-C18 in 76% yield. Next, thiol-ene click addition to endo-norbornene-cis-5,6-dicarboxylic acid was performed with the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.05% w/v), which proceeded Molecules 2023, 28, 1956 5 of 19 in an exo-selective manner, resulting in product 19. The desired product was purified with prep-C18 in 93% yield, and stereochemistry was indicated by 2D NOESY and HSQC NMR (supplied in supporting information, SI [25][26][27] and supported by the literature [33]. Asymmetric disulfide synthesis of 20 is a reversible conjugation to lactose-TEAB, where an unstable disulfide bridge is formed by an excess of dipyridyl disulfide reagent. The desired intermediate could be isolated and purified by prep-C18 but only in a modest yield (30%). Alternatively, the crude product can be used in situ to further react with a simple alkyl thiol, providing the disulfide 20 in 49% yield, once again after reverse-phase chromatography.
Halo acetamide addition to the lactose-TEAB proceeded in a similar fashion to the maleimide addition but with a longer time period of 2 h, resulting in 21 in 91% yield post-purification. The amino acid thioesterfication with the BOC-protected leucine was an important example of glycosylation of an amino acid, which proved to produce the stable thioester 22 in a moderate yield (50%, unoptimized) using N,N -dicyclohexylcarbodiimide (DCC, 2 eq.) and DMAP (0.1 eq.). Simple alkylation with an alkyl halide produced the thioether 23 slowly over 8 h. Oxidation of the thiol (14) could be observed, and inclusion of TCEP in the reaction conditions provided the desired product in 64% yield after purification. Finally, epoxide ring opening proceeded similarly to maleimide addition and acetamide alkylation, with a slightly shorter time of 1.5 h, giving product 24 in 75% yield post-purification. It is important to note that products 18, 21, and 24 can be obtained with minimal impurities or salts without C18 purification if a 1:1 stoichiometry, with respect to substrate and TEAB, is utilized. Overall, out of the seven reactions displayed, amines can only participate in two of the reactions and cannot form a reversible covalent bond, displaying a significant advantage thiol chemistry has over amines. Thiol-ene click chemistry is the most notable example of this, as the reaction is highly controlled via the presence of ultraviolet light and has been shown to be orthogonal to many other functional groups.
After demonstrating chemoselective conjugation with the thiol to a variety of coupling partners, the model lactose-TEAB probe (14) was applied to a more complex system to demonstrate the selectivity of a combined thiol-fluorophore-glycan. Upon reviewing the literature [25], bovine serum albumin (BSA) was chosen as a model protein partner to conjugate a glycan with since it possesses a singular reduced cysteine residue. Similar to the formation of compound 18, the thiolate ion present on BSA added to the PEGbismaleimide 25 [34]. After 1 h, the now linked BSA-PEG-maleimide was then submitted to dialysis in a 10-13 MWKO tube for 24 h to remove any small molecule reagents. The modified protein in solution was then reacted with a slight excess of lactose-TEAB (14) displayed in Figure 5 for an additional hour and finally purified using dialysis once again. After freeze-drying the sample, the identity of the glycoprotein 26 was confirmed using MALDI mass spectrometry (theoretical:~67,307 Da; observed mass: 67,309 Da), indicating a significant shift of~846 Da from the original BSA protein (66,463 Da). Analytical HPLC was used to track the reaction progress as well as the purity of the resultant product.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 20 75% yield post-purification. It is important to note that products 18, 21, and 24 can be obtained with minimal impurities or salts without C18 purification if a 1:1 stoichiometry, with respect to substrate and TEAB, is utilized. Overall, out of the seven reactions displayed, amines can only participate in two of the reactions and cannot form a reversible covalent bond, displaying a significant advantage thiol chemistry has over amines. Thiolene click chemistry is the most notable example of this, as the reaction is highly controlled via the presence of ultraviolet light and has been shown to be orthogonal to many other functional groups.
After demonstrating chemoselective conjugation with the thiol to a variety of coupling partners, the model lactose-TEAB probe (14) was applied to a more complex system to demonstrate the selectivity of a combined thiol-fluorophore-glycan. Upon reviewing the literature [25], bovine serum albumin (BSA) was chosen as a model protein partner to conjugate a glycan with since it possesses a singular reduced cysteine residue. Similar to the formation of compound 18, the thiolate ion present on BSA added to the PEG-bismaleimide 25 [34]. After 1 h, the now linked BSA-PEG-maleimide was then submitted to dialysis in a 10-13 MWKO tube for 24 h to remove any small molecule reagents. The modified protein in solution was then reacted with a slight excess of lactose-TEAB (14) displayed in Figure 5 for an additional hour and finally purified using dialysis once again. After freeze-drying the sample, the identity of the glycoprotein 26 was confirmed using MALDI mass spectrometry (theoretical: ~67,307 Da; observed mass: 67,309 Da), indicating a significant shift of ~846 Da from the original BSA protein (66,463 Da). Analytical HPLC was used to track the reaction progress as well as the purity of the resultant product. To further illustrate the importance of the soft nucleophilic nature of the thiol present on a fluorescent auxiliary, inspiration was taken from a 2-thiopyridine motif installed onto a Sepharose support [16], which was previously utilized as a method to purify proteins through covalent chromatography [12,35]. A commercially available thiol functionalized To further illustrate the importance of the soft nucleophilic nature of the thiol present on a fluorescent auxiliary, inspiration was taken from a 2-thiopyridine motif installed onto a Sepharose support [16], which was previously utilized as a method to purify proteins through covalent chromatography [12,35]. A commercially available thiol functionalized containing silica gel was determined as a direct way to obtain a 2-thiopyridine-activated support, and the thiol could readily be chlorinated [36] with sulfuryl chloride (10 eq.). The chlorinated thiols were then subsequently treated with 2-thiopyridine (2 eq.) in dry DCM, resulting in the desired thiol selective support (27). The activated SiliMet-SH (27,~0.31 mmol SH/g silica) was then applied to crude reductive amination reactions to investigate the effectiveness of a covalent chromatography method.
Following the process illustrated ( Figure 6), it was found that crude reactions could be purified through this method of washing and subsequent release with excess tributyl phosphine. The released glycans could then be precipitated, and any remaining organic compounds were removed via washing with dichloromethane. Most of the product loss was found to occur in the initial immobilization step, where the filtered solvent from the initial 'catch' was found to contain the functionalized glycan in the disulfide form (37-40% of original thiol by mass) along with 2-thipyridine. No significant amount of product in the reduced state thiol CH 2 at 2.70 ppm was observed in NMR analysis (of the initial filtrate), indicating complete capture of the desired thiol. Saturated solutions of ethylenediaminetetraacetic acid disodium salt and maltol, described in the literature [37], were applied to small portions of the activated support 27 as a method to remove redox catalyzing species, but the effect of both chelators on overall yield was found to be negligible. Overall, synthesis of other thiol-containing supports to improve activation or reduce the amount of redox-catalyzing species should be investigated to fully optimize this overall process. in the reduced state thiol CH2 at 2.70 ppm was observed in NMR analysis (of the initial filtrate), indicating complete capture of the desired thiol. Saturated solutions of ethylenediaminetetraacetic acid disodium salt and maltol, described in the literature [37], were applied to small portions of the activated support 27 as a method to remove redox catalyzing species, but the effect of both chelators on overall yield was found to be negligible.
Overall, synthesis of other thiol-containing supports to improve activation or reduce the amount of redox-catalyzing species should be investigated to fully optimize this overall process. Finally, to demonstrate that this auxiliary can facilitate glycoarray creation, the use of an appropriate alkene-coated surface was explored [38]. Thiol-coated slides were prepared through dip coating in 3-(Trimethoxysilyl)-1-propanethiol with HCl for 24 h. Iodine starch colorimetric titration was used to confirm the presence of thiols on the glass slide, and then they were further alkylated with a bis allyl PEG linker (28, 1 kg/mol) rather than the reported 1,2-polybutene (1.2 kg/mol) to avoid non-specific binding interactions to the surface [39]. The method of generating radicals onto the thiols was also optimized from the dip coating of slides for 24 h with UV irradiation to a drop casting approach with the radical initiator azobisisobutyronitrile (AIBN) in an air oven at 100 ⸰ C. Iodine starch titration was once again used to estimate the consumption of thiols to confirm that the thiol content had decreased by half, which indicated the presence of allyl-containing PEG on the drop-casted side of the slide.
The pegylated slides then underwent glycosylation, shown in Figure 7, where fluo- Finally, to demonstrate that this auxiliary can facilitate glycoarray creation, the use of an appropriate alkene-coated surface was explored [38]. Thiol-coated slides were prepared through dip coating in 3-(Trimethoxysilyl)-1-propanethiol with HCl for 24 h. Iodine starch colorimetric titration was used to confirm the presence of thiols on the glass slide, and then they were further alkylated with a bis allyl PEG linker (28, 1 kg/mol) rather than the reported 1,2-polybutene (1.2 kg/mol) to avoid non-specific binding interactions to the surface [39]. The method of generating radicals onto the thiols was also optimized from the dip coating of slides for 24 h with UV irradiation to a drop casting approach with the radical initiator azobisisobutyronitrile (AIBN) in an air oven at 100 • C. Iodine starch titration was once again used to estimate the consumption of thiols to confirm that the thiol content had decreased by half, which indicated the presence of allyl-containing PEG on the drop-casted side of the slide.
The pegylated slides then underwent glycosylation, shown in Figure 7, where fluorescence quantification of the TEAB fluorophore was measured, allowing for the optimization of various parameters, including lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP-0.1, 0.5, and 1% w/v), time of irradiation (1, 5, and 10 min), irradiation power (10, 20, and 40 mW/cm 2 ), and concentration of glycans (1, 5, and 10 mM), which were varied until optimized to the values given in Figure 7. Additionally, 1-[4-(2-hydroxyethoxy)-phenyl]-2hydroxy-2-methyl-1-propane-1-one (I2959), a popular photoinitiator, was also investigated but was found not to produce any fluorescence signal at similar concentrations to LAP, likely due to the poor molar absorptivity at the desired wavelength (365 nm) due to the overlap with the highly absorbing TEAB fluorophore. Utilizing the optimal glycosylation conditions, the quantifiable fluorescence of TEAB was then used to further optimize the PEGylation conditions, where the concentrations of PEG and AIBN (1, 5, 10, and 20% w/v) and the time (10, 15, 60, and 120 min) were varied. It is important to note that no significant fluorescence was observed when PEG and AIBN concentrations were dropped below 5% w/v, supporting the fact that thiol-ene addition was occurring on the alkene-PEG rather than disulfide formation to the SH-coated glass.  Figure 7. FITC concanavalin A binding assay of glycosylated PEG-coated slides in CFG notation, with error bars denoting 95% confidence intervals (n = 3). Blank is defined as a non-glycosylated region of the slide measured at the same time.
To fully establish that the surface was glycosylated, FITC-labeled concanavalin A from Canavalia ensiformis (1 mg/mL) was then used as a probe, as described in the literature [40], to investigate the binding of various glycans present on the surface. A slide of nine glycans was generated in triplicate spots, and a control region of PEG allyl-capped thiol was used to ensure sufficient washing of the surface. The variability in spotting, consistency of the surface, and washing of the FITC concanavalin A was optimized to minimize the variability of each well on the slide. Two standard deviations of the measure- Figure 7. FITC concanavalin A binding assay of glycosylated PEG-coated slides in CFG notation, with error bars denoting 95% confidence intervals (n = 3). Blank is defined as a non-glycosylated region of the slide measured at the same time.
To fully establish that the surface was glycosylated, FITC-labeled concanavalin A from Canavalia ensiformis (1 mg/mL) was then used as a probe, as described in the literature [40], to investigate the binding of various glycans present on the surface. A slide of nine glycans Molecules 2023, 28, 1956 8 of 19 was generated in triplicate spots, and a control region of PEG allyl-capped thiol was used to ensure sufficient washing of the surface. The variability in spotting, consistency of the surface, and washing of the FITC concanavalin A was optimized to minimize the variability of each well on the slide. Two standard deviations of the measurements at the blank region were determined to have contributed, on average, 51% (instrument error +/− 273 a.u.) of the error of the samples measured. Overall, the binding assay showed a significant signal for the primary binding target of concanavalin A, 1,3-α-1,6-α-D-mannotriose (15). The error was low enough to determine that there was a significant replicable binding event to 15 relative to a non-binding monosaccharide such as D-ribose-TEAB (11) as well as partial binding to a variety of other glucose-bearing glycans which have also been reported to bind display partial binding to concanavalin A in other research [38]. Mass spectrometry data were collected through the University of Illinois School of Chemical Sciences' mass spectrometry service. ESI was collected on a Waters Quattro Ultima mass spectrometer, and MALDI analysis was performed on a Bruker Autoflex Speed LRF MALDI. UV-Vis and fluorescence data were acquired on a SpectraMax i3x reader (San Jose, CA, USA), and UV irradiation was performed with an OmniCure S2000 Spot UV curing system (Saint Louis, MO, USA). Optical rotation was measured on a Jasco DIP-370 digital polarimeter (Easton, MD, USA).
Compound previously published in the literature [28]. (4) N,N -(dithiodi-2,1-ethanediyl)bis[2-amino-benzamide] 3 (100 mg, 0.256 mmol, 1 eq.), TCEP (367 mg,1.28 mmol, 5 eq.), and Chelex 100 sodium form (1 g) were suspended in MeOH (10 mL). The suspension was stirred for half an hour; then, the Chelex was filtered off and the filtrate was concentrated to dryness. The crude product was re-dissolved in water (10 mL), and the desired product was extracted with ethyl acetate (50 mL three times). The resulting organic layer was washed with a saturated NaCl solution (10 mL five times), and the resulting organic layer was concentrated, yielding the colorless oil 4 (38 mg, 37% yield). 1  Compound previously published in the literature [28]. (5) N,N -(dithiodi-2,1-ethanediyl)bis[2-amino-benzamide] 3 (500 mg, 1.282 mmol, 1 eq.) and zinc powder (420 mg, 6.462 mmol, 5 eq.) were suspended in MeOH, water, and acetic acid (25 mL, 3.5:3.5:3). The suspension was heated at 65 • C for 2.5 h and then left to cool. The excess undissolved metallic zinc was then filtered off from the solution using P2 filter paper. The crude mixture was then concentrated under vacuum to remove most of the methanol and water until a fine oil formed at the bottom of the flask. The product was then precipitated with an excess of water, and the solid was filtered with P2 filter paper. The white precipitate was once again washed with water, finally yielding pure zinc salt 5 (547 mg, 94% yield). 1

General Synthesis of Glyco-Conjugates (6-17)
A glycan of interest (1.0 eq.), Zn-TEAB 5 (1.0 eq.), 2-picoline borane (1.2 eq.), and EDTA (2.5 eq.) were dissolved in a MeOH, water, and acetic acid mixture (3.5:3.5:3, [glycan] = 0. 1 M). The solution was heated to 65 • C and stirred for 2.5 h. The reaction was then filtered through a cotton plug to remove the undissolved EDTA. All reactions were then concentrated under vacuum, filtered through a 0.45 µM filter, and purified by reverse-phase chromatography on a semi-preparative C18 column. All desired glycans were eluted with a gradient of 99:1% to 50:50% (water: acetonitrile) over a 15-min period. If the resulting product contained trace amounts of fluorophore due to partial co-elution, the starting material could be extracted away with a large excess of ethyl acetate with the product fully dissolved in water. All glyco-conjugates should be stored as a solid with no solvent present in a −20 • C freezer to avoid oxidation if storing for longer than a week.
3.4.9. Synthesis of Lactose-TEAB-PEG-Bismaleimide BSA (26) BSA (10 mg, 0.000150 mmol, 1 eq.) and N,N -[1,2-ethanediylbis(oxy-2,1-ethanediyl)] bismaleimide 25 (2.3 mg, 0.00750 mmol, 50 eq.) were dissolved in water (1 mL), and the solution was left to stir for 1 h. The solution then underwent dialysis with 10-13 MWKO tubing against water (3.5 L), with the dialysate being changed every 12 h for 24 h. Lactose-TEAB 14 (0.4 mg, 0.000766 mmol, 5.1 eq.) was then added to the solution and left to stir for an additional hour. The solution underwent dialysis again, was placed inside of a 10-13 MWKO tubing, and was left to undergo dialysis against water (3.5 L) for a further 3 days, with the dialysate being changed every 12 h. The product was then lyophilized, yielding the synthetic BSA glycoprotein 26 (10 mg) in excellent yield. The final product was analyzed via MALDI to determine connectivity to the desired lactose-TEABmaleimide connector.
3.5. Application of TEAB Glycans 3.5.1. Preparation of Activated SiliMet-SH (27) SO 2 Cl 2 (25 g, mmol, 10 eq.) was poured into a round-bottom flask, and SiliMet-SH (12.500 g, 19.25 mmol SH content from supplier, 1 eq.) was slowly added to the fuming mixture over a period of 5 min. Once completed, the silica gel was placed onto a rotary evaporator and heated to 45 • C, and reduced pressure was applied until the complete removal of any excess SO 2 Cl 2 . Separately, 2-thiopyridine (4.280 g, 38.5 mmol, 2 eq.) was dissolved in dry dichloromethane (150 mL) and slowly added to the dried chlorinated silica. The sample was stirred for 10 min and then filtered through a glass sinter, and the resin was washed with dichloromethane, methanol, and acetone. The silica was then further washed with sodium bicarbonate (20 mL) to quench any unreacted chlorine present, re-acidified with 1M HCl (40 mL), and further washed with water (200 mL) until the run-off was between pH 6 and 7. Finally, the activated silica was dried with acetone (200 mL) and dried further under vacuum overnight, yielding the purified activated SiliMet-SH (27,14.678 g).

Optimized Catch and Release Process
To the solution of recently reductively aminated glycans, activated resin (~0.31 mmol of SH/g of resin) was added and stirred for 2 h. The solution was diluted with water and then centrifuged for 3000 rpm at 5 min, which was repeated with methanol and then acetone. Following this, the resin was recovered and suspended in PBu 3 (10 eq.) with ethanol:water (1:1), where it was stirred for 30 min and the solution was separated from the resin via a syringe filter (1 µM); the process was repeated an additional two times. The resin was further washed with additional water and methanol; then, the filtrates combined to be concentrated under vacuum. Next, the yellow crude oil was precipitated with dichloromethane and centrifuged at 3000 rpm for 15 min, and the supernatant was filtered through a syringe filter (1 µM) which was then discarded; additional dichloromethane was added to the pellet, and the process was repeated a further two times. Finally, the precipitate was dissolved in a minimal amount of water and syringe-filtered (1 µM) into a scintillation vial, to which the solution was concentrated, yielding the desired product.

Example Catch and Release of Acarbose-TEAB
Following the optimized procedure, a crude reaction of the reductively aminated acarbose-TEAB (16, theoretical yield: 64 mg, 0.0776 mmol, 1 eq.) was added to the activated SiliMet-SH (250 mg). The suspension was left to stir for 2 h and was then placed into a centrifuge tube, diluted up to 15 mL with water, and centrifuged at 3000 rpm for 5 min. The supernatant was decanted, and the process was repeated with the same volume of methanol and acetone. The resin was recovered back into a scintillation vial, then, PBu 3 (157 mg, 0.775 mmol, 10 eq.) was added and diluted with water:ethanol (1:1, 5 mL). The suspension was stirred vigorously for 30 min, and the solution was back-filtered with a PTFE syringe filter (1 µM); this process was repeated 3 times, and the resin was washed with water (10 mL) and methanol (10 mL), followed by pooling the filtrates together and concentrating until dry. The resulting yellow oil was then diluted with dichloromethane (50 mL) and once again centrifuged at 3000 rpm for 15 min. The supernatant was then discarded, additional dichloromethane (30 mL) was added to the pellet, and the process was repeated a further two times. The pellet was dissolved in water (5 mL x 3) and filtered through a PTFE syringe filter (1 µM). The filtered solution was dispensed into a scintillation vial, which was then concentrated under rotary evaporation to yield the desired acarbose-TEAB (16, 37 mg, 0.0450 mmol) in 58% yield.

Preparation of Bis Allylated PEG 1000 (28)
Following the literature [41], PEG 1000 MW (10.000 g, 10 mmol, 1 eq.) was added to a 3-necked round-bottom flask and flushed with nitrogen, to which THF (100 mL) was added and stirred until dissolved. To the stirring solution, NaH (60% in oil, 1.3 g, 55 mmol, 5 eq.) was slowly added; then, allylbromide (8.64 mL, 100 mmol, 10 eq.) was added dropwise. The reaction was heated at 40 • C, with additional portions of NaH (5 eq.) being added every 30 min until a total of 15 eq. was reached. After 12 h, the solution was left to cool and then concentrated to obtain the crude product, which was then re-dissolved in DCM (30 mL) and filtered. Again, the sample was concentrated to dryness, and the crude solid was then washed with hexanes (200 mL) three times to remove mineral oil leftover from the NaH, yielding the diallyl PEG 1000 MW (8.734 g, 7.74 mmol,~95% purity) in 77% yield with trace mineral oil by NMR. and dichloromethane again. Next, the slides were dried under vacuum and a portion was taken, and the thiol content was estimated utilizing the iodine and starch colorimetric indicator, which approximated around~3.6 µmol/mm 2 . A saturated solution of divinyl PEG 1000 MW (10% w/v) and AIBN (10% w/v) in toluene (9.23 mL) was then prepared, and a portion of the solution (1 mL) was deposited onto the thiol glass slide in a culture dish. The slide was 'baked' in an air oven at 100 • C for 1 h and then removed and sequentially washed with an excess of toluene, dichloromethane, methanol, water, and acetone. The now pegylated slides were then utilized for glycosylation, and the free thiol content was estimated to have decreased to~1.6 µmol/mm 2 with the iodine starch colorimetric assay.

Glycosylation of PEG-Coated Microscope Slides
To the surface of the pegylated slides, a solution of the glycan of interest (10 mM) and LAP (1% w/v) in water was dispensed (2 µL). The solution was exposed to UV light (365 nm, 10 mW/cm 2 ) for a period of 1 min and was then washed away with excess water, methanol, and acetone, leaving fluorescent spots when viewed with a UV flashlight (365nm). The slide was then dried under vacuum, resulting in the prepared glycosylated surface.

Conclusions
TEAB is a promising glycan auxiliary, where the use of reductive amination with a glycan has allowed for the preparation of thiol-functionalized glycans in 60-90% yield utilizing standard C18 chromatography, unlike amino derivatives. In addition, TEAB has been shown to undergo rapid conjugation chemoselectively with a variety of coupling partners in 49-93% yield, whilst amino/amide derivatives cannot undergo many of these reactions. These versatile reaction pathways demonstrate the benefits of TEAB as a glycan auxiliary in glycomics investigations. Preliminarily, TEAB-conjugated glycans can be used in glycoprotein synthesis by utilizing reduced cysteine residues. Initial testing of a 2-thiopyridine-activated support demonstrated the utility of a thiol conjugate, where mg quantities of the labeled glycans could be recovered from crude mixtures without the need for chromatographic separations, but this must be validated on reductively aminated biological glycan sources and with confirmation of C18 retention. Additionally, preliminary photolithography of the glycan conjugates demonstrated that glycan arrays can be generated and measured to determine binding to primary enzymatic substrates. Future studies should assess the effect of glycosylation on proteins with respect to folding, substrate binding, and protein-protein interactions. Finally, with respect to photolithography, many applications could be permitted to allow for studies of glycans to biological targets. However, more research surrounding the enzymatic compatibility (glycosyltransferases) to TEAB glycans, assessments of how applicable the pull-down approach is on biological samples, and more in-depth surface characterization (morphology, roughness, and spacing from the surface) of the alkene-pegylated slides are still required. Overall, this novel glycan auxiliary proved to be a diverse, bifunctional, and cost-efficient target to accommodate various studies surrounding the glycome, expanding on previous amino-based derivatives.